1. Field of the Invention
The present invention relates to a novel amino acid derivative, N.sup..alpha. -tert-butoxycarbonyl-N.sup..epsilon. -(N-bromoacetyl-.beta.-alanyl)-L-lysine (BBAL), for use in solid-phase or solution-phase peptide synthesis for introducing a side-chain bromoacetyl group at any desired position in a peptide sequence. The bromoacetyl group is subsequently used for the preparation of cyclic peptides, peptide conjugates and peptide polymers. Such peptide derivatives are useful in preparing potential peptide immunogens, vaccines and therapeutics, and for substances such as peptides linked to polymers, plastics, enamels and ceramics.
2. Background Art
Conjugates of synthetic peptides with proteins, other peptides, polymers (soluble/insoluble; natural/synthetic), and surfaces of special materials are being employed increasingly in biomedical research and biotechnology. Applications of peptide conjugates include the preparation of immunogens (including synthetic vaccines) for raising antibodies to selected portions of protein antigens (Lerner, R. A. (1982) Tapping the immunological repertoire to produce antibodies of predetermined specificity. Nature 299, 592-596; Zavala, F. et al, (1985) Rationale for development of a synthetic vaccine against Plasmodium falciparum malaria. Science 228, 1436-1440; Tam, J. P. (1988) Synthetic peptide vaccine design: Synthesis and properties of a high-density multiple antigenic peptide system. Proc. Natl. Acad. Sci. U.S.A. 85, 5409-5413; and Milich, D. R. (1989) Synthetic T and B cell recognition sites. Adv. Immunol 45, 195-282), affinity adsorbents, immunoassay components, and cell adhesion surfaces (Massia, S. P. et al, Covalent surface immobilization of Arg-Gly-Asp- and Tyr-Ile-Gly-Ser-Arg-containing peptides to obtain well-defined cell-adhesive substrates. Anal. Biochem. 187, 292-301; and Brandley, B. K. et al, (1988) Covalent attachment of an Arg-Gly-Asp sequence to derivatizable polyacrylamide surfaces: Support of fibroblast adhesion and long-term growth. Anal. Biochem. 172, 270-278).
The locus of attachment of a peptide to its conjugate partner may have a major influence on the desired biological activity or performance of the conjugate (Dyrberg, T. et al, (1986) Peptides as antigens. Importance of Orientation. J. Exp. Med. 164,1344-1349; Schaaper, W. M. M. et al, (1989) Manipulation of antipeptide immune response by varying the coupling of the peptide with the carrier protein. Mol. Immunol. 26, 81-85; and Golvano, J. et al, (1990) Polarity of immunogens: Implications for vaccine design. Eur. Immunol. 20, 2363-2366). Strategies for cross-linking a peptide through a single, selected locus are often complicated by the presence of more than one amino or carboxyl group, and the need to protect (then deprotect) amino groups if a carboxyl function is to be activated. Peptides can be more or less selectively derivatized through their N-terminal amino groups by means of acylation reactions in slightly acidic media and/or with use of certain types of reagents, such as, symmetrical anhydrides (10). However, if one wishes to synthesize a peptide in order to prepare a conjugate, planning the synthesis for this purpose can prove very advantageous. For example, some workers have introduced a reactive cysteine residue at the desired position for heteroligation (Green, N. et al, (1982) Immunogenic structure of the influenza virus hemagglutinin. Cell 28, 477-487 and Bernatowicz, M. S. et al, (1986) Preparation of peptide-protein immunogens using N-succinimidyl bromoacetate as a heterobifunctional crosslinking reagent. Anal. Biochem. 155, 95-102); Drijfhout et al completed a sequence on a solid support with an N-terminal S-acetylmercaptoacetyl group (Drijfhout, J. W., Bloemhoff et al, (1990) Solid-phase synthesis and applications of N-(S-acetylmercaptoacetyl) peptides. Anal. Biochem. 187, 349-354). The deprotected peptide was treated with hydroxylamine to remove the S-acetyl group and then joined through its N-terminal sulfhydryl group to a conjugate partner bearing an SH-selective electrophilic function (e.g., an N-substituted maleimide or an .alpha.-haloacetyl moiety) by means of very stable thioether cross-links (Bernatowicz, M. S. et al (1986), Anal. Biochem. 155, 95-102, supra and Drijfhout, J. W. et al (1990), supra).
Strategy considerations may give preference to placing sulfhydryl groups on the conjugate partner (or using those already present) and coupling it to Cys peptides by means of less stable disulfide bonds. The coupling can be effected through an activating group, such as S-(3-nitro-2-pyridinesulfenyl)(Npys), previously placed on a cysteine side chain (Bernatowicz, M. S. et al, (1986) Preparation of Boc-[S-(3-nitro-2-pyridinesulfenyl)]-cysteine and its use for unsymmetrical disulfide bond formation. Int. J. Peptide Protein Res. 28, 107-112; Drijfhout, J. W. et al, (1988) Controlled peptide-protein conjugation by means of 3-nitro-2-pyridinesulfenyl protection-activation. Int. J. Peptide Protein Res. 32,161-166; and Ponsati, B. et al, (1989) A synthetic strategy for simultaneous purification-conjugation of antigenic peptides. Anal. Biochem. 181, 389-395). A Cys(Npys) residue, introduced in solid-phase peptide synthesis (SPPS), will remain intact during trifluoroacetic acid (TFA) or HF cleavage steps.
For an alternative approach, the peptide synthesis program could be modified in order to introduce an SH-selective electrophile somewhere in the sequence, again allowing use of stable thioether cross-linkages. Lindner and Robey, F. A., (1987) Automated synthesis and use of N-chloroacetyl-modified peptides for the preparation of synthetic peptide polymers and peptide-protein immunogens. Int. J. Peptide Protein Res. 30, 794-800) described the incorporation of N-terminal chloroacetyl-glycylglycyl groups in the last cycle of an automated SPPS. Subsequently, Robey, F. A. and Fields, R. L. (Robey, F. A. et al, (1989) Automated synthesis of N-bromoacetyl-modified peptides for the preparation of synthetic peptide polymers, peptide-protein conjugates, and cyclic peptides. Anal. Biochem. 177, 373-377) and Kolodny, N. and Robey, F. A. (Kolodny, N. et al, (1990) Conjugation of synthetic peptides to proteins: Quantitation from S-carboxymethylcysteine released upon acid hydrolysis. Anal. Biochem. 187, 136-140) described a similar method for introducing the more reactive bromoacetyl group at the N-termini of peptides made by SPPS. The former method is also described in U.S. patent application Ser. No. 07/283,849 (filed Dec. 3, 1988). Using this approach, these authors have prepared many useful immunogens either as peptide-protein conjugates or as self-polymers of peptides that contain both a cysteine residue and an N-terminal bromoacetyl group.
The main limitation to the above approach is lack of flexibility in choosing the site for an a-haloacetyl group. To our knowledge, there has not yet been reported a method for introducing an SH-selective alkylating function at any desired position in a peptide that is being synthesized sequentially.
Accordingly, it is desired to obtain a method for introducing a bromoacetyl or chloroacetyl or other haloacetyl or haloacyl cross-linking function at the N -or C-terminus or at any intermediate position of a synthetic peptide that is being prepared.